CN88102435A - Albuminous assay method in the biofluid - Google Patents
Albuminous assay method in the biofluid Download PDFInfo
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- CN88102435A CN88102435A CN198888102435A CN88102435A CN88102435A CN 88102435 A CN88102435 A CN 88102435A CN 198888102435 A CN198888102435 A CN 198888102435A CN 88102435 A CN88102435 A CN 88102435A CN 88102435 A CN88102435 A CN 88102435A
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- reagent
- reaction
- damping fluid
- speed
- protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
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- Urology & Nephrology (AREA)
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- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
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- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
Serum and the reaction of dinitro halobenzene compound, and measure the speed that produces halogen.Albumin content is in direct ratio in this speed and the sample.Other protein do not disturb this reaction as globulin and free amino acid and urea.
Description
This method the invention relates to albuminous method in the quantitative measurement serum, even also can be measured in the presence of globulin and other haemocyanins.
Albuminous technology is important on medicine, diagnosis and clinical chemistry in mensuration blood and the biofluid (for example urine).Generally, total protein is determined by so-called " biuret/copper reaction " in the serum.This reaction relates to Cu
2+And the band look complex (purple) that forms between the peptide bond of protein.When measuring albumin and globulin respectively, with globulin precipitation, and centrifuging is also measured albumin in the supernatant separately with biuret reaction then with salting out method (for example with alkali or ammonium sulfate).Because during this operating cost, method improvement is the direct albumin in the mensuration serum in the presence of other protein are arranged now.For example Delaney is in 1964 (Proc.Australian Assoc.Clin.Biochem.64(1964), 1) introduced the method quantitative measurement seralbumin of bromine cresols edge (BCG) dyestuff combination.The serum BCG(pH7.0 of buffering) dilution, and measure its absorbance in wavelength 615nm and reduce, absorbance is to be linear change along with albumin concentration (up to 50mg/ml), and other do not disturb with the protein of electrophoretic separation such as haemoglobin and cholerythrin.
Yet preferably can find operation more specifically, that is to say to find out more not to be vulnerable to the method that other molecules disturb in the serum.The method that is summarised in claim 1 (is a sero-abluminous method in a kind of quantitative measurement biofluid (even having globulin to be present in wherein), it is characterized in that: (1) blood serum sample and excessive halo-aromatics reagent reacting, wherein have at least a halogen atom to be replaced, and this reaction cause the formation of protein/reagent complex by protein; (2) measure this reaction rate; (3) it is calculated that the speed data that records with other isotopic numbers, these coordination data are to finish in same reaction with the albumin calibrator quantity, provide the said determination result by calculating) be to reach this purpose important step.This is complete beyond thought discovery, can quantitatively determine albumin by this approach, even has a large amount of globulin existence also can determine down.In fact, applicable that class reagent of the present invention, for example adjacent, to the dinitro halobenzene, known be can usefulness protein NH
2One end group carries out bimolecular nucleophilic displacement of fluorine (seeing Sager Biochem.J.39(1945), 507,1945), its speed order be F＞Cl ~ Br. this be beyond thought, at least when being fluorine, halogen shows that albumin is actually unique protein type that can produce significant reaction speed in serum, even this reaction rate is at other protein, for example globulin also can produce under existing.
Another beyond thought discovery is must not come assaying reaction speed with the optics of record formation complex or the variation of other character, thereby and can measure fluor releasing quantity and learn reaction rate easily.This is beyond thought, though because above-mentioned reaction (seeing following reaction equation) comprises following fluorine metalepsis (X represents fluorine), do not show obviously that one finds the mode (for example measuring with the fluoride electrode of ion selectivity) that can directly measure surely.Reaction equation is as follows:
A kind of reasonable reagent is can a small amount of water-soluble dinitrofluorobenzene (DNFB) compound.So in order to obtain best result, the available suitable aqueous medium through buffering compatible with blood serum sample comes emulsification DNFB organic solution.For example, DNFB in benzene solution and by the emulsification of buffer substance institute (characteristics are 0.1～1N solution of pH5.5～7.7), and the blood serum sample that will analyze stirs with emulsion fluid.The fluoride of separating out is used the fluorine ion selective electrode determining of any usual type again.Calibration curve is to obtain by one group of albumin standard solution, and the sample and the calibration curve contrast of unknown concentration are measured.Be present in the globulin of normal concentration scope in the serum and other haemproteins and free amino acid and urea and do not produce interference.
Organic reagent DNFB, ether, toluene, DMF(dimethyl formamide), the DMSO(dimethyl sulfoxide (DMSO)), the THF(tetrahydrofuran), EtOH(ethanol) and MeOH(methyl alcohol) also suitable, but less usefulness is because some can play nucleation and make drift electrode among them.
The selection of buffer substance is also very important, find, for example Guan Yong acetate, trishydroxymethylaminomethane and dimethyl arsenic acid buffer liquid, its pH value is in above-mentioned pH value scope, the result produces drift electrode, does not then see drift electrode with phosphate buffer (pH5.8～7.6).Though the reason of these difference is not also studied, can suppose that tentatively the organic buffer material plays nucleophilic some halogen atoms are replaced.Though this method can be operated (time that is consecutive steps is controlled, and makes measurement result that reappearance be arranged) with the organic buffer liquid that can make electrode produce drift, also can use inorganic damping fluid, for example phosphate is the optimized buffer material.
Below commutable step successfully be used for replacing providing reaction medium with aqueous buffer solution emulsification DNFB reagent.
With filter paper of DNFB solution impregnation and allow its drying.
When analyzing, impregnated dry filter paper is placed beaker bottom, add working buffer solution and low amount of fluorinated thing, stir with magnetic stirrer, electrode immerses solution, adds blood serum sample after a period of stabilisation, and measures its reaction rate.
Usually obtain in the memory storage that this reaction rate is stored in microprocessor (this device is connected with the instrument of habitually practising potential electrode) by known albumin solution, when measuring unknown sample content, with the speed record, and calculate the automatic result of albumin concentration in the sampling with the data of storage.This result may be displayed on graduated recording chart or is presented on the video screen that naked eyes can see.
Below with example in detail the present invention.
Fig. 1 is a calibration graph, this diagram shows in phosphate buffer, the F during pH7.6
-Produce the funtcional relationship of rate variations and albumin concentration.
Fig. 2 is similar to Fig. 1, is to carry out when pH5.8 but measure.
Example 1
Draw the 5M solution of 0.1ml DNFB in benzene with pipette, drop on Whatman (whatman) filter paper of diameter 2cm, and circular filter paper is dry in stream of warm air.Prepare and store several this circular filter papers before use.
Be formulated in the 0.1M phosphate buffer, the albumin calibration sample of pH7.6, its scope is 10～100mg/ml.
With polypropylene beaker (10ml) and F
-Electrodes selective (the Orion compound electrode is connected on Orion 901 ion analysers that are connected with Apple II E microcomputer) is measured.
Gan Zao filter paper places beaker (10ml) bottom, and with 5ml phosphate buffer and 5ml10
-3The MNaF aqueous solution is incorporated in together in the beaker and (stable current potential baseline is provided and can returns baseline rapidly after this to mensuration).Solution stirs with magnetic stirrer, and electrode is immersed in the solution, allows it stablize 1～2 minute.Add a five equilibrium (0.1ml) standard solution then, the F of generation
-Be that current potential (mV) change records by ion-selective electrode is measured.Measurement result is recorded on the curve of Fig. 2, shown in the abscissa is albuminous concentration (mg/ml) wherein, and shown in the ordinate is time dependent F
-Concentration ((d ［ F
-])/(dt), μ Mol/l/min＜micromoles per liter/minute), this variation is to be obtained by the rate curve of trying to achieve with the method for Nernst equation formula (NERNST equation).
Also carry out same experiment in the presence of the globulin of difference amount, experiment shows that the globulin up to 40mg/ml does not have interference effect yet, and this concentration is higher than the globulin concentration that can run in the serum far away.
Measure the blood serum sample of unknown albumin concentration and contrast the check of Fig. 1 curve with said method.The gained result is very consistent with the result that method with prior art records.
Example 2
Repetition is still operated in phosphate buffer (pH5.8) in the experiment of example 1 explanation.The pH value of serum keeps the pH value identical with example 1.The result that Fig. 2 curve marks, similar to the result that Fig. 1 marks basically, but its speed is lower.Carry out the sample that method for measuring is equally applicable to unknown content at pH5.8, but its sensitivity is lower.Have interference measurements not of globulin and other haemproteins, free amino acid and urea.
Except DNFB, having other reagent of similarity also can use aspect the release fluorine, the red sulfuryl fluoride that for example is expressed from the next:
Following compound also can use:
2,4, the derivant that 6-three halos symmetry triazine class and one or two halogen atom thereof are replaced by organic substituent, and 2,2,3,3-ptfe ring butane-1-formamide.Their structural formula is respectively:
Wherein Hal represents fluorine, chlorine and bromine; R is an organic group.
Claims (8)
1, sero-abluminous method in a kind of quantitative measurement biofluid (even having globulin to be present in wherein), it is characterized in that: (1) blood serum sample and excessive halo-aromatics reagent reacting, wherein have at least a halogen atom to be replaced, and this reaction cause the formation of protein/reagent complex by protein; (2) measure this reaction rate; (3) it is calculated that the speed data that records with other isotopic numbers, these coordination data are to finish in same reaction with the albumin calibrator quantity, provide the said determination result by calculating.
2, be dinitrofluorobenzene (DNFB) or red sulfuryl fluoride according to the reagent that the process of claim 1 wherein.
3, cause halogen to replace according to the reaction that the process of claim 1 wherein, and the speed of measuring is the speed that halide ion discharges.
4, according to the method for claim 3, wherein halogenide is with F
-The fluoride that electrodes selective is measured.
5, according to the method for claim 4, reaction medium wherein is the damping fluid of pH5.5～7.8.
6, according to the method for claim 4, damping fluid inorganic-containing compound wherein, for example phosphate, and pH value is 5.8～7.6.
7, according to the method for claim 5, reagent wherein is to be dissolved in the organic reagent, and this kind of equal portions solution is with above-mentioned damping fluid emulsification.
8, according to the method for claim 5, reagent wherein is to be evenly distributed on a slice porosint, and after this, this porosint contacts with sample in damping fluid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP87810253A EP0287745B1 (en) | 1987-04-22 | 1987-04-22 | Method for the determination of albumin in biological fluids |
| EP87810253.2 | 1987-04-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN88102435A true CN88102435A (en) | 1988-11-09 |
Family
ID=8198398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198888102435A Pending CN88102435A (en) | 1987-04-22 | 1988-04-22 | Albuminous assay method in the biofluid |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US5110746A (en) |
| EP (1) | EP0287745B1 (en) |
| KR (1) | KR890700831A (en) |
| CN (1) | CN88102435A (en) |
| AT (1) | ATE69509T1 (en) |
| AU (1) | AU607767B2 (en) |
| DE (1) | DE3774570D1 (en) |
| ES (1) | ES2028124T3 (en) |
| GR (1) | GR3003619T3 (en) |
| WO (1) | WO1988008533A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0399127A1 (en) * | 1989-05-23 | 1990-11-28 | Pharmacia ENI Diagnostics Inc. | Homogeneous immunochemical method for determining haptens by means of ion selective electrodes |
| ATA194989A (en) * | 1989-08-17 | 1990-04-15 | Koller Ernst | METHOD FOR DETECTING AND DETERMINING HUMAN SERUM ALBUMINE IN BIOLOGICAL LIQUIDS WITH THE AID OF FLUOROGENIC CYANINE DYES |
| AU609630B1 (en) * | 1989-08-30 | 1991-05-02 | Miles Inc. | Method and composition for the assay of albumin |
| GB9216582D0 (en) * | 1992-08-05 | 1992-09-23 | Bristol Polytechnic | Determination of enzyme activity |
| EP1459059A4 (en) * | 2001-11-26 | 2005-01-19 | Ischemia Tech Inc | Electrochemical detection of ischemia |
| GB2435328B (en) * | 2006-02-15 | 2010-04-28 | Inverness Medical Switzerland | Methods and device for indirectly detecting the presence of an analyte |
| KR102205101B1 (en) | 2014-02-28 | 2021-01-19 | 닛토덴코 가부시키가이샤 | Urinalysis device and Dry Reagent for Quantitative Urinalysis |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE577056A (en) * | 1956-05-21 | |||
| GB1267186A (en) * | 1970-02-20 | 1972-03-15 | Warner Lambert Pharmaceutical | |
| DE2510633C3 (en) * | 1975-03-12 | 1978-07-13 | Boehringer Mannheim Gmbh, 6800 Mannheim | Diagnostic agent for the detection of protein in body fluids and indicator dyes suitable therefor |
| US4118194A (en) * | 1977-04-08 | 1978-10-03 | Rockwell International Corporation | Sensor for fluid components |
| JPS5819222B2 (en) * | 1977-06-21 | 1983-04-16 | 森永乳業株式会社 | Method for producing steroid-serum albumin complex sensitized latex particles |
| US4330296A (en) * | 1980-06-23 | 1982-05-18 | Beckman Instruments, Inc. | Albumin reagent and assay |
| GB2118304B (en) * | 1982-03-22 | 1986-01-15 | Sira Ltd | Detecting surface deviations |
| JPS58179359A (en) * | 1982-04-14 | 1983-10-20 | Fuji Photo Film Co Ltd | Multilayer analyzing material for quantitative determination of protein |
| US4568647A (en) * | 1983-10-11 | 1986-02-04 | Eastman Kodak Company | Method and element for albumin assay |
| US4670218A (en) * | 1984-02-24 | 1987-06-02 | Miles Laboratories, Inc. | Ion test means having a porous carrier matrix |
-
1987
- 1987-04-22 EP EP87810253A patent/EP0287745B1/en not_active Expired - Lifetime
- 1987-04-22 DE DE87810253T patent/DE3774570D1/de not_active Expired - Fee Related
- 1987-04-22 ES ES198787810253T patent/ES2028124T3/en not_active Expired - Lifetime
- 1987-04-22 AT AT87810253T patent/ATE69509T1/en not_active IP Right Cessation
-
1988
- 1988-04-22 CN CN198888102435A patent/CN88102435A/en active Pending
- 1988-04-22 AU AU17185/88A patent/AU607767B2/en not_active Ceased
- 1988-04-22 US US07/299,939 patent/US5110746A/en not_active Expired - Fee Related
- 1988-04-22 WO PCT/US1988/001328 patent/WO1988008533A1/en unknown
- 1988-12-21 KR KR1019880701698A patent/KR890700831A/en not_active Application Discontinuation
-
1992
- 1992-01-22 GR GR920400061T patent/GR3003619T3/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ES2028124T3 (en) | 1992-07-01 |
| KR890700831A (en) | 1989-04-27 |
| US5110746A (en) | 1992-05-05 |
| AU1718588A (en) | 1988-12-02 |
| GR3003619T3 (en) | 1993-03-16 |
| ATE69509T1 (en) | 1991-11-15 |
| WO1988008533A1 (en) | 1988-11-03 |
| DE3774570D1 (en) | 1991-12-19 |
| EP0287745B1 (en) | 1991-11-13 |
| EP0287745A1 (en) | 1988-10-26 |
| AU607767B2 (en) | 1991-03-14 |
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| PB01 | Publication | ||
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| SE01 | Entry into force of request for substantive examination | ||
| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |